Ultrasensitive electrochemical biosensors

ABSTRACT

An electrochemical biosensor includes a working electrode modified with a redox polymer and amine-terminated capture aptamer specific for a particular detection target. The binding sequence of the capture aptamer is also complementary to part of a second ssDNA which is labeled with HRP (horseradish peroxidase). The capture aptamer will form dsDNA with the HRP-labeled ssDNA and bring HRP into electrical contact with the redox polymer and the electrode. Prior to capturing the detection target, addition of H 2 O 2  will lead to the highest reduction current due to the redox polymer-mediated, enzyme-amplified electroreduction of H 2 O 2 .

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Award Number NIHULIGM118973 awarded by the National Institutes of Health. The U.S.government has certain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to biosensors and methods for their use.

SUMMARY OF THE INVENTION

According to the present invention, there is presented the use of redoxpolymers such as PVI-Os(bpy)₂ to carry reducible/oxidizable groups suchas Os(bpy)₂ complexes on their flexible side chains and the use of redoxpolymers in conjunction with redox enzymes and aptamers to achieve thedevelopment of ultra-sensitive electrochemical biosensors.

More specifically, a working electrode in an electrochemical cell ismodified with the redox polymer by electro-crosslinking/depositingPVI-Os(bpy)₂, amine-terminated capture aptamer (specific for aparticular detection target is co-deposited with the redox polymer. Partof the capture aptamer (the binding sequence) is also complementary topart of a second ssDNA which is labeled with HRP (horseradishperoxidase), and the capture aptamer will form dsDNA with theHRP-labeled ssDNA and bring HRP into electrical contact with the redoxpolymer and the electrode. Prior to capturing the detection target,addition of H₂O₂ will lead to the highest reduction current due to theredox polymer-mediated, enzyme-amplified electroreduction of H₂O₂.

Alternatively, the electrode can be modified with the redox polymerhydrogel, PVI-Os(bpy)₂, using a crosslinker such as poly(ethyleneglycol) diglycidyl ether; i.e., PEGDGE. It is then followed byelectrodeposition of amine-terminated capture aptamer.

The PVI-Os modified aptamer-sensing electrode (made withelectrodeposition of PVI-Os or PESGDGE crosslinking) is immersed in abuffer solution (pH 7.4) containing the aptamer, and a negativepotential (e.g. −1.4 V) (vs. Ag/AgCl) is applied for a certain amount oftime, which allows the aptamer to be immobilized in the PVI-Os film.Then the electrode is immersed in a buffer solution containingHRP-labeled ssDNA which forms double helix with the immobilized captureaptamer. The electrode is then labeled with the enzyme HRP whichcatalyzes the electroreduction to H₂O₂ to water. A reduction current canbe detected when H₂O₂ is present in the solution.

The effectiveness of the invention was demonstrated with detection ofaflatoxin by immersing the above electrode sensor in a solutioncontaining aflatoxins and H₂O₂. Binding of aflatoxin by the captureaptamer released some of the HRP-labeled ssDNA, leading to loweredreduction current which served as a measure of the level of aflatoxin.See FIG. 1.

The redox polymer/aptamer/redox enzyme-modified electrode sensor of theinvention is not limited to aflatoxins; by changing the aptamer, theelectrode can be made sensitive to virtually any kind of toxins,biological molecules, even viruses and bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of a redox polymer/aptamer/HRP basedaflatoxin sensor and assay according to an embodiment of the invention.

FIG. 2 shows a scheme for the preparation of poly (1-vinyl imidazole)“PVI” according to an embodiment of the invention.

FIG. 3 shows a reaction for forming an osmium complex according to anembodiment of the invention.

FIG. 4 shows a chemical reaction of PVI with OS(bpy)₂Cl^(+/2+) accordingto an embodiment of the invention.

FIG. 5 shows the structure of Os(bpy)₂Cl complexed withpoly(1-vinylimidazole) according to an embodiment of the invention.

FIG. 6 is a representation of dialysis of PVI-Os complex according to anembodiment of the invention.

FIG. 7 is a representation of an electrodeposition process according toan embodiment of the invention.

FIG. 8 is a representation of the electrodeposition of a PVI-Os complexaccording to an embodiment of the invention.

FIG. 9 shows the chemical structure of PEGDGE.

FIG. 10 shows crosslinking between PVI and PEGDGE according to anembodiment of the invention.

FIG. 11 shows an EC cell schematic according to an embodiment of theinvention.

FIG. 12 is a chart showing current versus voltage for electrodepositionaccording to an embodiment of the invention.

FIG. 13 is a chart showing scan rate vs. peak current forelectrodeposition according to an embodiment of the invention.

FIG. 14 is a chart showing current versus voltage for crosslinkingaccording to an embodiment of the invention.

FIG. 15 is a chart showing peak current versus the square root of scanrate during crosslinking according to an embodiment of the invention.

FIG. 16 shows the results from detection of aflatoxin AFB₁ using abiosensor according to an embodiment of the invention.

DETAILED DESCRIPTION

Poly (1-vinyl imidazole) (“PVI”) and osmium bipyridine complexes areprepared separately. Then, they are chemically derived into theOsmium-PVI (Nakabayashi et. al, 2001) polymer. Next, the chemical sensorelectrode is made also using known methods from literature (Ohara et.al, 1993) and modified with catechol cross-linked with chitosan (Zhanget. al, 2012). The hydrogel PVI_(n)-Os can function as a high conductorof electrons into the modified chemical sensor electrode. PVI_(n)-Ospolymers are highly water-soluble with redox potential of up to 200 mV.The voltammogram of this polymer has constant ΔEp (separation of peaks)value around 100 mV which shows that the electron diffusion kinetics isbetter than other similar films. These polymers form amperometricglucose sensors by linking with glucose oxidase (GOX). The procedures tomake the above-mentioned chemicals are described in detail below.

Preparation of poly (1-vinyl imidazole)

Poly (1-vinyl imidazole) forms a group of hydrogels which have anadequate electron diffusion coefficient. This is used with an enzymaticelectrode which can then conduct electrons through the gel's polymernetwork to the electrodes. In the preparation of PVI, the polymerizationof 1-vinyl imidazole involves the bulk thermal polymerization using 2,2′-azobis-isobutyronitrile (AIBN) as a radical initiator. For thispurpose, a mixture containing 3 ml of 1-vinyl imidazole and 0.156 gramsof AIBN is heated at 70° C. for one hour under an inert atmosphere ofN₂. The inert atmosphere may be created by flushing a gentle stream ofN₂ that displaces the oxygen existing in air. Such inert atmosphere iscrucial to remove any traces of oxygen, which would otherwise react withexisting radicals, inducing termination of the bulk radicalpolymerization process. FIG. 2 shows the formation structure of PVI.

After heating is complete, a dark yellow precipitate of PVI is obtainedwith a yield of about 65.7%.

Purification of PVI

The synthesized polymer, once cooled down to room temperature, isdissolved in methanol and recrystallized by adding the solution dropwiseto acetone while stirring vigorously with a magnetic bar. The solutionis then filtered using vacuum filtration. The obtained final polymer, ofa pale yellowish color, is purified PVI with a yield of about 34.4%.

Preparation of the Osmium Bipyridine Complex

The preparation of the osmium bipyridine complex may be carried outusing known methods. The osmium (II) complex with PP (bpy, dmbpy),imidazole and Cl⁻ can act as a much more efficient electron transfermediators for glucose sensors.

Commercial (NH₄) OsCl₆ is heated with bipyridine at reflux for 1 hourusing 1,2-ethanediol medium under an inert atmosphere and continuousmagnetic stirring. 0.5 g of (NH₄) OsCl₆ is mixed with 0.356 g of PP (bpyin this case). The heat level is preferably kept at 9. The continuousstir level is preferably kept at 3. The reaction mixture is then allowedto cool down at room temperature. The cooled mixture is then treatedwith solution of 5.2 g of Na₂S₂O₄ and 30 ml of water to reduce anyosmium bipyridine ions that may have formed in the reaction mixture. Theprecipitate of dark violet-black color is collected after cooling for 30min in ice bath. The associated chemical reaction is shown in FIG. 3.

The osmium bipyridine complex precipitate may be further purified usingvacuum filtration. The solid may be washed with water and then diethylether.

Alternative Method (Microwave) of Cis-[OsCl₂(PP)₂] Synthesis

According to an alternative method, the osmium bipyridine complex may bemade according to a microwave method using 0.2 g of (NH₄) OsCl₆, 0.1424g of PP and 4 ml of 1,2-ethanediol in a microwave container. The mixturemay be heated using microwave at 200° C. for 20 min. The reactionmixture is allowed to cool down at room temperature afterwards. Then thecooled reaction mixture is treated with a solution of 2.6 g Na₂S₂O₄ and15 ml of water to reduce any osmium bipyridine ions that may have formedin the reaction mixture. The precipitate of dark violet-black color iscollected after cooling for 30 min in ice bath. The precipitate may bepurified by washing with water and diethyl ether. The cycle voltammetryΔEp values for Os (II) complex are typically 56-65 mV indicating fastheterogeneous electron transfer potential in the complex. After the PVIand Os complex has been formed, the cross linking of Os-PVI is conductedusing chemical derivatization of the redox polymer.

Preparation of the Chemically Derivatized Os-PVI Polymer

After filtration, the obtained polymer is subjected to chemicalderivatization through the bonding of osmium. The chemicalderivatization process involves the reaction of the synthesized polymerwith Os(bpy)₂Cl₂. Briefly, this method involves the preparation of amixture of 0.13 g of the vinyl imidazole polymer with a solution of 0.2g of Os(bpy)₂Cl₂ in 10 ml ethanol and heating at reflux conditions for72 hours. The mass required for PVI is calculated with molarcalculations. The osmium bipyridine complex has the chemical formula ofC₂₀H₁₆Cl₂N₄O₅. The molecular weight is 573.506 g/mol. The number ofmoles required for 0.2 g of Os(bpy)₂Cl₂ to react with the PVI, thenumber of moles for osmium complex can was obtained as follows:

${n = {\frac{0.2}{57{3.5}06} = {{0.000343873 \times 4} = {{0.0}014\mspace{14mu}{mol}}}}},$

where N=4 (no. of molecules of compound). Therefore,m=n×M=0.0014×94=0.13 g of PVI.

The mixture is heated at 45° C. for three days. The chemical reaction isshown in FIG. 4.

Following the heating, the reflux is complete, and the osmium bipyridineis complexed with the PVI. The final complex is shown in FIG. 5.

The reflux may also be carried out using microwave assisted organicsynthesis. In this case the mixture of 0.1 g of Os(bpy)₂Cl₂ and 0.065PVI with 4 ml of ethanol is heated at 120° C. for one hour in themicroSYNTH microwave system, significantly shortening the reflux time.

Dialysis of PVI-Os Complex

Dialysis may be performed for purification purposes to remove unwantedchemicals. This can also be performed to purify the PVI-Os complex. Ifperformed, the following steps may be followed with reference to FIG. 6:

-   -   Add 800 ml of water to a 1000 mL beaker.    -   Place 25 mL of PVI-Os in dialysis tubing and leave in the beaker        overnight.    -   Replace the water and repeat four times, optionally using        new/clean beakers.

Polishing Electrode

For the purposes of conducting CV studies, a properly polished electrodeis essential. The electron transfer should not be interfered by thecontaminants present in the electrode surface. The electrode may bepolished according to any known method. Mechanical polishing may becarried out as follows:

-   -   Add 0.3 micron of alumina powder (Al₂O₃) to glass and then add        water.    -   Polish the electrodes using circular    -   Rinse off the electrode after polishing it with the alumina        solution.    -   Keep the electrode in a vial that contains water that has been        sonicated.    -   Let the vial (with electrode) sonicate for 2 min in a sonicator.    -   Take out the electrode and rinse off the electrode

After performing the steps described above, the electrodes can be usedin their respective places in the electrochemical cell as workingelectrode and reference electrode.

Electrodeposition of PVI-Os Complex to the Electrode

The process of electrodeposition on the electrode depends on the scanrate. Higher the current, the higher the scan rate which is shown below.

Current∝Scan rate

Current∝√{square root over (Scan rate)}

The redox polymer can be electrodeposited using known electrodepositionmethods. During electrodeposition, the current arranges random movingPVI-Os molecules on surface bound electrode in the presence of electronsand chloride ions as shown in FIG. 7. The chemical reaction for electrodeposition is shown in FIG. 8.

Preparation of the Chemical Sensor Electrode

According to an alternative method, the redox polymer can be crosslinkedusing a PEGDGE crosslinker. The electrode is then modified with thecrosslinked polymer which forms a hydrogel in solution. This includesadding the PVI-Os complex to Poly (ethylene glycol) diglycidyl ether,i.e., PEGDGE together in solution. The PEGDGE structure (Peg 400) isshown in FIG. 9.

The detailed procedures is as follows:

-   -   2.5 mL of PEGDGE is added to a vial;    -   1 mL of deionized (DI) water was added to the above solution;    -   100 mL from solution above is added to a new vial with 200 mL        PVI-Os1 solution (i.e., 5 mL PVI-Os1 and 1 mL buffer of pH 7);    -   7.5 mL is taken from the second vial and added on the electrode;    -   The mixture is left to cure overnight to make the first layer of        the electrode. To prepare the sensor electrode, first the        electrode was modified by crosslinking PVI-Os with PEGDGE, see        FIG. 10.

An example of an EC cell is shown in FIG. 11. The PVI-Os modifiedelectrode is immersed in a PBS (pH 7.4) solution containing 10 μM of aselected detection aptamer, and a negative potential of −1.4 V (vs.Ag/AgCl) is applied for 10 min which allows the aptamer to beimmobilized in the PVI-Os film. Then the electrode is ready for use inthe EC cell as a biosensor using the electrolyte solution and selectedanalytes as desired. Aptamers are single-stranded oligonucleotides thatcan strongly and selectively bind to target molecules. The aptamer-basedbiosensors of the present invention may use uses enzyme-linked aptameror fully synthesized oligonucleotides, for example, in the case for AFB₁detection.

Testing of Biosensor

The effectiveness of the biosensor of the present invention wasdemonstrated by detection of aflatoxin AFB₁. The aptamer and HRP enzymeoligonucleotide were ordered from Integrated DNA Technologies (Iowa,USA). The sequence of the DNA-HRP that was used was/5HRPMD/AAAAAATGTGGGCCTAGCGA (SEQ.ID.No. 1) in an amount of 250 nmol.DNA-Amino (aptamer) was ordered in 2 batches of 1 μmole and 5 μmole withsequence /5AmMC12/AGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCAC A(SEQ.ID.No. 2) as ordered. The yield guarantee was different for thesetwo batches with the first being 44 nmol and later being 219.8 nmol. Thebiosensor conditions were varied with different electrodes. Thetemperature was kept constant at 37° C. In the procedures for testing AFwith the biosensor, only the amounts of AF were changed whereas theaptamer and DNA-HRP was kept constant. The following were the generalprocedures that were followed during testing of AF:

The respective electrode was crosslinked using PVI-Os and PEGDGE asdescribed above. The crosslinked electrode was also tested using PBSusing CV.

The aptamer that was obtained from external source was electrodepositedonto the electrode. This was also tested with CV and I-t studies. Thevoltage was kept at −1.4 V- and two-time conditions were applied: 5 minand 10 min. Later, the electrode was rinsed with water and tested withbuffer two times using CV as well.

Incubation of HRP, AFB₁ and PBS was done so that reaction could takeplace in solution. The electrode was kept inside the incubated solutionfor 20 min at 37° C. and tested again. Only the amounts of AFB₁ werechanged for the several test runs. The electrode was tested for CV at0.6-0.2 V range. The amount of AFB₁ that was used during the experimentsranged from 0.1 ng/mL to 100 ng/mL.

The electrode from the above step was put into H₂O₂ solution to measurethe AF level. Also, CV test and I-t test were conducted. The reason forusing H₂O₂ solution is that it leads to the highest reduction of currentmaking the detection of AF faster.

Experiments utilizing four different electrodes (labeled) and AFB₁quantities are shown in table below:

TABLE 1 Selected experimental runs Electrode HRP-DNA (μM) AFB₁ (ng/mL)C1 1 μM None C2 None None C3 1 μM  0.1 ng/mL C4 1 μM 0.01 ng/mL

During the testing, the aptamer quantity was kept constant at 10 μM andH₂O₂ was kept at 1 mM.

Results and Discussion

All electrochemical studies were performed using a CH InstrumentElectrochemical analyzer in a single compartmental cell with athree-electrode configuration comprising a Pt wire as the auxiliaryelectrode, a glassy carbon electrode as the working electrode, andAg/AgCl as the reference electrode. The CV profiles were obtained forthe electrodeposition process, cross-linking and rhenium compoundsinteraction with DNA.

Data from Electrodeposition

During the electrodeposition of the redox polymer, several scan rateswere utilized to obtain the voltammograms. There is dependence of thescan rate of the cyclic voltammograms with that of the completedelectrodeposited polymer as scan rates were varied from 50 mV/s to 1000mV/s. Results are shown in FIG. 12.

It can be seen that as the scan rate increases so does the peak current;however, the peak separation remains almost constant, indicating areversible process.

As shown in FIG. 13, peak current is directly proportional to the scanrate, suggesting that the redox species is surface-bound and that theredox process is diffusion-independent.

Data from Cross Linking

The cross linking of the redox polymer via PEGDGE forms the electricalwire with which the electrons can be transferred from the targetmolecule with help of the solvent in the EC cell. This way there ishigher sensitivity and freedom for the electrons to move. The CV graphat FIG. 14 shows asymmetrical voltammograms, but the peaks of separationhave higher values compared to the electrodeposition results.

As can be seen in FIG. 14, with PEGDGE crosslinking, the peak currentincreases drastically with increasing scan rate; furthermore, the peakcurrent is proportional to the square root of the scan rate (FIG. 15),suggesting this is diffusion controlled, according to the Randles-Sevcikequation:

$i_{p} = {0.4463{{nFAC}\left( \frac{nFvD}{RT} \right)}^{1/2}}$

where,

-   -   i_(p)=current maximum in amps    -   n=number of electrons transferred in the redox event (usually 1)    -   A=electrode area in cm²    -   F=Faraday Constant in C mol⁻¹    -   D=diffusion coefficient in cm²/s    -   C=concentration in mol/cm³    -   v=scan rate in V/s    -   R=Gas constant in J K⁻¹ mol⁻¹    -   T=temperature in K

or,

i _(p)=(268,600)n ^(3/2) AD ^(1/2) Cv ^(1/2)

at 25° C. Although PVI-Os is crosslinked and deposited on the electrodesurface, the redox species, the Os complex, still behaves like diffusedspecies in a solution, unlike the case of electrodeposition where the Oscomplex behaves as surface-confined species. The reason for this is thatwith PEGDGE crosslinking PVI-Os forms a flexible and gigantic matrix,and upon hydration forms a micro-environment that is very similar to thebulk solution; Os complex inside the matrix therefore behave asdiffusional species and follows the Randles-Sevcik equation. This alsoexplains the excellent electron-conducting property of thePEGDGE-crosslinked PVI-Os polymer.

Data from AFB₁ Tests

The last phase of testing consisted of testing aflatoxin AFB₁ in thepresence of aptamer and redox enzyme (HRP), the results of which areshown in FIG. 16. There were varying conditions used in which theconcentration of the aflatoxins was changed to create some optimalconditions for binding the target molecules. The CV and I-t testsconducted for these varying conditions. These conditions are thepresence (in different quantities of AFB₁) and absence of it from thesolution. The sensor electrode (PVI-Os/aptamer-modified electrode) wasincubated in 300-4, PBS (pH 7.4) solution containing 1 μM HRP-oligo andvaried amounts of AFB₁ at 37° C. for 20 min.

The electrode was then rinsed with PBS and immersed in PBS containing 1mM H₂O₂, and while a potential of 0.1 V was applied the reductioncurrent was recorded as shown in the above figure. The stabilizedcurrent (which should be obtained in about 3 min) was then used as ameasure of the concentration of AFB₁ in the sample. The backgroundcurrent was obtained using the sensor electrode (PVI-Os/aptamer-modifiedelectrode) without the incubation step. Since no HRP-DNA was present tobe captured by the aptamer, H₂O₂ would not be reduced at the electrodehence the detected current was extremely low, ˜5.5×10⁻⁸ A. The detectionwas based on a competitive model in which the immobilized aptamer couldbind to both the HRP-oligo and AFB₁. However, the binding between theaptamer and AFB₁ is stronger than that between the aptamer and theHRP-oligo. When there was no AFB₁ in the sample the amount ofaptamer-bound HRP-oligo was the most, resulting in the highest reductioncurrent of H₂O₂ (˜8.8×10⁻⁶ A). As the concentration of AFB₁ increased inthe sample, less HRP-oligo would be captured by the aptamer, leading todecreased reduction current. It can be clearly seen from the figure thathigher amounts of AFB₁ reduces the current generated from the analytesolution.

Comparing the working range of the tests conducted we can see from thedata that anywhere from 0.01 ng/mL to 0.1 ng/mL of sensitivity of AFB₁samples can be detected using this novel method. In literature, workingsensitivity level as low as 0.05 ng/mL has been reported for AFB₁ oncorn and paprika using extraction methods based in methanol-PBS andmethanol-water, respectively. Therefore, the novel electrochemicalimmunosensor for AFB₁ that is described here has higher sensitivitylevels than previous biosensors. The novel method takes advantage of theaptamer-PBS solution to keep the detection of AFB₁ to low levels as 0.01ng/mL.

Biosensors were constructed using both electrodeposition and PEGDGEcrosslinking. PEGDGE crosslinking resulted in sensors with largerreduction current and higher sensitivity.

As shown in FIG. 16, when the AFB₁ concentration decreases in thesample, the reduction current of H₂O₂ increases as more HRP-labeled DNAget captured by the aptamer. The highest reduction current is, ofcourse, 8.8×10⁻⁶ A when there is no AFB₁ in the sample. A 0.01 ng/mLconcentration of AFB₁ is readily detected, which is comparable or betterthan most current detection methods. Furthermore, at 0.01 ng/mL thereduction current is only ˜3×10⁻⁶ A, leaving a very broad range from8.8×10⁻⁶ A (maximum current). We expect the detection limit to besignificantly lower than 0.01 ng/mL.

1. A method for the detection of biological targets, comprisingproviding a biosensor comprising an electrochemical cell comprising areference electrode, a working electrode, and a counter electrode in ashared volume, the working electrode having fixed to its surface a redoxpolymer, immobilizing a target-specific capture aptamer on the workingelectrode; adding an electrolyte solution into the shared volume;applying a voltage across the working electrode and the counterelectrode; adding to the electrolyte solution in the shared volume asample solution suspected of containing the biological target and asolution containing an HRP-labeled oligonucleotide having a nucleotidesequence that is complimentary to a sequence of the capture aptamer;measuring a reduction current in the electrochemical cell.
 2. The methodof claim 1, wherein the redox polymer is Osmium derivitizedpoly(1-vinylimidazole).
 3. The method of claim 1, wherein the redoxpolymer is electrodeposited on the working electrode.
 4. The method ofclaim 1, wherein the redox polymer is crosslinked to the workingelectrode with PEGDGE.
 5. The method of claim 1, wherein the workingelectrode is a carbon-based electrode, the counter electrode is aplatinum wire, and the reference electrode is Ag/AgCl electrode.
 6. Themethod of claim 1, wherein the nucleotide sequence of the HRP-labeledoligonucleotide is complimentary to a sequence of the biological target.7. The method of claim 1, wherein the electrolyte is H₂O₂.
 8. The methodof claim 7, wherein the HRP catalyzes the electroreduction of the H₂O₂to water.
 9. The method of claim 1, wherein the biological target isAFB₁.
 10. The method of claim 1, wherein the HRP-labeled oligonucleotidecomprises a nucleotide sequence of SEQ.ID.No.
 1. 11. The method of claim1, wherein the target-specific capture aptamer comprises a nucleotidesequence of SEQ.ID.No. 2
 12. A biosensor comprising: a referenceelectrode, a working electrode, and a counter electrode in a sharedvolume, the working electrode having fixed to its surface a redoxpolymer, a target-specific capture aptamer immobilized on the workingelectrode; an electrolyte solution in the shared volume and incommunication with the reference electrode, the working electrode andthe counter electrode; a voltage source for applying a voltage acrossthe working electrode and the counter electrode; a solution containingan HRP-labeled oligonucleotide having a nucleotide sequence that iscomplimentary to a sequence of the biological target.
 13. The biosensorof claim 12, wherein the redox polymer is Osmium derivitizedpoly(1-vinylimidazole).
 14. The biosensor of claim 12, wherein the redoxpolymer is electrodeposited on the working electrode.
 15. The biosensorof claim 12, wherein the redox polymer is crosslinked to the workingelectrode with PEGDGE.
 16. The biosensor of claim 12, wherein theworking electrode is a carbon-based electrode, the counter electrode isa platinum wire, and the reference electrode is Ag/AgCl electrode. 17.The biosensor of claim 12, wherein the nucleotide sequence of theHRP-labeled oligonucleotide is complimentary to a sequence of thebiological target.
 18. The biosensor of claim 12, wherein theelectrolyte is H₂O₂.
 19. The biosensor of claim 12, wherein the HRPcatalyzes the electroreduction of the H₂O₂ to water.
 20. The biosensorof claim 12, wherein the biological target is AFB₁.
 21. The biosensor ofclaim 12, wherein the HRP-labeled oligonucleotide comprises a nucleotidesequence of SEQ.ID.No.
 1. 22. The biosensor of claim 12, wherein thetarget-specific capture aptamer comprises a nucleotide sequence ofSEQ.ID.No. 2.